In prokaryotes, DNA replication proceeds bidirectionally from a single specific initiation site. In eukaryotes DNA replication is bidirectional from multiple initiation sites. We intend to determine whether eukaryotic initiation sites are specific and whether their temporal sequence of activation is constant by preparing a replication map of a few yeast chromosomal DNA's and large restriction endonuclease fragments of chromosomal DNA's. By hybridizing pulse-labelled DNA from very early S-phase cells to cloned yeast DNA fragments we should be able to identify fragments containing initiation sequences and further characterize the sequences. We will isolate yeast mitochondrial DNA and analyze its structure and replication intermediates by gradient techniques and electron microscopic observations. We will determine how the amount of mitochondrial DNA per cell is regulated by perturbing the normal amount and analyzing the return to normal.